It is known that the acetylation of phosphoric acid by the use of an acetylating agent such as acetic anhydride or ketene can give mixtures of monoacetyl phosphate and diacetyl phosphate, occasionally further containing triacetyl phosphate. A crystalline salt of monoacetyl phosphate to be used in the above enzymatic reaction can be separated from these mixtures. Representative methods include a process for the preparation of diammonium acetyl phosphate which comprises acetylating 100% phosphoric acid with acetic anhydride in ethyl acetate and pouring the reaction mixture into chilled methanol saturated with ammonia (G. M. Whitesides et al., J. Org. Chem. 44, 864). However, this method involves an operation at low temperatures, i.e. -30 TO -10.degree. C., and the formed diammonium acetyl phosphate tends to decompose in the presence of excess ammonia, so that the reaction conditions must be strictly controlled in order to carry out the separation by filtration smoothly. Alternatively, salts of acetyl phosphate can be obtained in the form of an aqueous solution from the above acetylation reaction mixture (D. C. Crans and G. M. Whitesides, J. Org. Chem. 48, 3130-32), but the product is inconvenient for the storage or the use as compared with the solid one, because acetyl phosphate hydrolyses in an aqueous solution.
It is also known that a salt of phosphoric acid is acetylated with acetic anhydride to obtain a salt of acetyl phosphate, as described in West German Patent No. 2831831 or East German Pat. No. 144546. In these patents, acetic anhydride or its mixture with acetic acid was used as a medium, and the examples disclosed in these patents demonstrated that when the acetylation of sodium or potassium phosphate was carried out in acetic anhydride or its mixture with acetic acid in which sodium or potassium phosphate can not be dissolved, the reaction proceeded to the stage of diacetyl phosphate. Accordingly, as described in the latter half of the East German Patent, solvolysis with alcohol etc. was necessary to obtain monoacetyl phosphate from the diacetyl derivative. Up to this time, the acetylation of lithium phosphate was disclosed only in the West German Patent No. 2831831. According to Example 1 of this patent, a solution of lithium hydroxide (12 parts or 0.5 molar part) in acetic anhydride (408 parts or 4 molar parts) is added dropwise to a mixture of 100% phosphoric acid (49 parts or 0.5 molar part) and acetic anhydride (204 parts or 2 molar parts) in a nitrogen atmosphere under stirring, and the resulting mixture is further stirred at 40.degree. to 50.degree. C. for 2 hours to obtain a pale yellow, transparent reaction mixture. This mixture is evaporated to dryness under a reduced pressure at 65.degree. C. by the use of a rotary evaporator to obtain a white solid residue (93 parts). All analyses such as elemental analysis, ignition test, acetyl group determination or titration with potassium hydroxide ensure that this residue is lithium diacetylphosphate.
As described above, the acetylation of lithium phosphate with acetic anhydride according to the prior art proceeds easily to the diacetyl product stage, and gives a homogeneous solution.
Lithium monoacetyl phosphate itself, which is selected as a salt suitable for obtaining acetyl phosphate in the form of a solid salt in the present invention, is already well known in the purification and isolation of monoacetyl phosphate from an aqueous solution (see E. R. Stadtman and F. Lipmann, J. Biol. Chem., 185, 549-551; Methods in Enzymology, vol. III, pp. 228-231, ed. S. P. Colowick and N. 0. Kaplan (1957); D. E. Koshland, Jr., J. Amer. Chem. Soc., 73, 4103; A. W. D. Avison, J. Chem. Soc., 1955, 736). These separation methods are based on the property of dilithium monoacetylphosphate that it is hardly soluble in a mixture of ethanol and water.
According to R. W. Porter et al. (J. Biol. Chem., 244, 1847) dilithium acetyl phosphate was precipitated by addition of methanol containing lithium acetate to an oily, evaporation residue which was obtained by treating monotriethylammonium salt of phosphoric acid, (C.sub.2 H.sub.5).sub.3 NH H.sub.2 PO.sub.4), with acetic anhydride in acetonitrile for one hour at room temperature followed by removing volatile materials using a rotary evaporator.
As described above, the medium which has been used in the prior art to precipitate a lithium salt of acetyl phosphate is an alcoholic solvent which is reactive to acetic anhydride. Accordingly, the precipitation of the lithium salt could not be directly combined with the acetylation using acetic anhydride.
As described above, there is not known as yet a simple process for precipitating a crystalline salt of monoacetyl phosphate which can be easily handled from the acetylation product of phosphoric acid without recourse to a low temperature operation. Further, the acetylation of phosphate salts proceeds without control to the diacetyl product stage, and otherwise in order to precipitate a lithium salt of monoacetyl phosphate the replacement of the medium of acetylation by another medium was necessary.